US20080269469A1 - Method of Separating Protein - Google Patents

Method of Separating Protein Download PDF

Info

Publication number
US20080269469A1
US20080269469A1 US10/594,441 US59444104A US2008269469A1 US 20080269469 A1 US20080269469 A1 US 20080269469A1 US 59444104 A US59444104 A US 59444104A US 2008269469 A1 US2008269469 A1 US 2008269469A1
Authority
US
United States
Prior art keywords
condition
target protein
isoelectric point
ion
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/594,441
Other languages
English (en)
Inventor
Hiroshi Yanagisawa
Yoko Sakakibara
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of US20080269469A1 publication Critical patent/US20080269469A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography

Definitions

  • the invention relates to a method of separating proteins taking advantage of their isoelectric points and applications of the method.
  • Salting-out, gel filtration (molecular sieve) and various chromatographic techniques taking advantage of physical and chemical properties of the substance to be purified have been usually combined for separating and purifying proteins and glycoproteins, and affinity chromatography using lectins has been particularly considered to be effective for purifying glycoproteins.
  • the yield of the substance to be extracted may be lowered or deterioration of the extracted substance may occur depending on the separation and purification method, and the procedure of the method may be complicated.
  • lectins should be selected for respective types of the sugar chain of the glycoprotein in advance in the affinity chromatography that has been thought to be effective for purification of the glycoprotein, and discrimination of the type of the sugar chain of the glycoprotein to be separated is inevitable. It is also a defect of this method that the cost for separation and purification is expensive.
  • the objective of the invention is to solve at least one of the above-mentioned problems.
  • Isoelectric point chromatography takes advantage of isoelectric points of proteins comprising the steps of adsorbing a target protein on a support, and eluting the target protein. It has been a common method in conventional isoelectric point chromatography to adsorb the protein on the support at a low ion strength, and elute the protein by increasing the ion strength.
  • a target substance can be adsorbed as the ion strength is lower, it is also possible that substances other than the target substance are also adsorbed.
  • the inventors of the invention have paid attention to the above-mentioned problems, and have made intensive studies on the conditions for separating the glycoprotein. As a result, it was found that the glycoprotein in the sample could be selectively separated and recovered under conditions contradictory to conventional common knowledge, namely the adsorption is performed at high ion strength, and elution is performed at low ion strength. It was confirmed from further studies that glycoproteins having weak binding ability to the support could be separated and recovered by lowering the ion strength for adsorption.
  • the separation method having the above-mentioned features may be advantageous for the separation and recovery of the glycoprotein, it was also suggested that the method may be also applicable to simple and efficient separation and purification of ordinary proteins, as well as the glycoprotein, depending on the adsorption conditions and supports to be used.
  • a method for separating proteins comprising the steps of:
  • a method for separating proteins comprising the steps of:
  • step (c) The method for separating proteins according to (5), wherein step (c) is applied under substantially the same condition as the first condition.
  • the pH in the first condition is lower than the isoelectric point of the target protein
  • the ion exchanger is a cation exchanger
  • the pH in the second condition is in the vicinity of or higher than the isoelectric point of the target protein.
  • the pH in the first condition is higher than the isoelectric point of the target protein
  • the ion exchanger is an anion exchanger
  • the pH in the second condition is in the vicinity of or lower than the isoelectric point of the target protein.
  • the sample contains a plurality of target proteins
  • step (b) comprises the step of continuously eluting the proteins under a solvent condition corresponding to the isoelectric points of respective target proteins.
  • Adsorption of unnecessary substances to a support can be efficiently prevented in the method according to the invention since adsorption (and washing) is performed under a condition at high ion strength.
  • a condition for lowering the ion strength for elution is effective for specifically eluting the target protein by preventing elution of the unnecessary substances due to increased ion strength. According to the invention employing such condition, it is possible to separate and purify the target protein by a simple operation within a short period of time.
  • FIG. 1 shows the result (chromatogram) of purification of pea diamine oxidase by the method according to the invention.
  • FIG. 2 is a table showing the yield of diamine oxidase purified by the method of the invention.
  • the lower table shows the yield when diamine oxidase is purified by a conventional method (Purification and Characterization of Amine Oxidase from Pea Seedlings, F. Vianello, A. Malek-Mirzayans, M. Luisa Di Paolo, R. Stevanata, and A Rigo, Protein Express and Purification 15, 196-201 (1999)).
  • FIG. 3 shows the result (chromatogram) of purification of egg white by the method according to the invention.
  • FIG. 4 shows the result of electrophoresis of the final product of egg white purified by the method according to the invention.
  • FIG. 5 shows the result (chromatogram) of egg white purified by the method according to the invention.
  • FIG. 6 shows the result of electrophoresis of the final product of egg white purified by the method according to the invention.
  • FIG. 7 shows the results (chromatograms) of separation of egg white albumin, transferrin and streptavidin by the method according to the invention using CM Sephadex as an ion exchanger.
  • FIG. 8 shows the results (chromatograms) of separation of egg white albumin, transferrin and streptavidin by the method according to the invention using CM Sepharose as an ion exchanger.
  • FIG. 9 shows the results (chromatograms) of separation of egg white albumin, transferrin and streptavidin by the method according to the invention using SP Sephadex as an ion exchanger.
  • FIG. 10 shows the results (chromatograms) of separation of egg white albumin, transferrin and streptavidin by the method according to the invention using SP Sepharose as an ion exchanger.
  • FIG. 11 shows the result of chromatography adsorbed under a condition of higher ion strength (0.2 M tris-succinate buffer).
  • FIG. 12 shows the result of chromatography washed with a high concentration of a buffer solution.
  • FIG. 13 shows a result of separation (chromatogram) of carbonic anhydrase and peroxidase by the method according to the invention.
  • FIG. 14 shows the result (chromatogram) of separation of all the proteins loaded on the column using the tris-acetic acid buffer solution.
  • FIG. 15 shows the result (chromatogram) of separation of all the proteins loaded on the column using the tris-citric acid buffer solution.
  • the invention relates to a method for separating proteins, and comprised following steps (a) and (b) in the first aspect:
  • the protein as a target of separation (also referred to the “target protein” in the specification) is not particularly limited to in the invention.
  • the target is preferably a protein having sugar chains, or a glycoprotein.
  • the term “protein” in the specification represents a concept including the glycoprotein, except that the term is clearly described in contrast to the glycoprotein.
  • proteins as the target of separation in the invention include egg white albumin, diamine oxidase and transferrin. According to the invention as shown in examples below, separation was successful in all the attempts of separation of egg white albumin, diamine oxidase and transferrin. Accordingly, the invention is applicable for a variety of glycoproteins.
  • separation refers to separation of the target protein from a sample
  • purification refers to separation of the target protein from a sample
  • the invention may be used for separating only the target protein from a sample containing plural kinds of proteins (including a case where a part of them is glycoproteins).
  • the plural kinds of proteins may be separated by one operation.
  • the plural kinds of the proteins serve as the target proteins in this case.
  • the invention is particularly favorable for separating specified proteins or glycoproteins in the sample containing proteins and glycoproteins together. Specifically, when proteins and glycoproteins having sugar chains added thereto are present together in the sample, the invention may be utilized for separating either of them.
  • Step (a) is performed under a condition (first condition) at high ion strength and at a pH outside of the vicinity of the isoelectric point of the target protein.
  • the sample contacts an ion exchanger typically by adding the sample to a column packed with the ion exchanger in this step.
  • the sample is usually added to the column equilibrated with an appropriate buffer solution in advance.
  • a continuous operation from adsorption to elution may be facilitated by employing such mode of column chromatography.
  • the sample may be directly added to the ion exchanger not packed in a specified vessel such as a column in order to permit the target protein to contact the ion exchanger.
  • Such batch type treatment method is advantageous for facilitating a large quantity of the sample to be treated in one operation.
  • Step (a) is performed at high ion strength. It was revealed by the inventors that adsorption at high ion strength prevents foreign substances from being adsorbed, or the target protein in the sample can be specifically adsorbed. Proteins were adsorbed at low ion strength by using, for example, a buffer solution with a concentration of 20 mM in conventional isoelectric point chromatography. However, one feature of the invention is that proteins are adsorbed at relatively higher ion strength than conventional conditions by using, for example, a tris-succinate buffer solution with a concentration of as high as 0.1 M, instead of adsorbing at above-mentioned low ion strength.
  • high ion strength refers to, for example, a concentration of about 0.05 M or more, preferably about 0.075 M or more, more preferably about 0.1 M or more, and further preferably about 0.2 M or more when a buffer solution comprising a combination of a weak acid and weak base is used, although the concentration differs depending on the kind of the target protein and the kind of the sample containing the protein, and on the kind of the buffer solution used.
  • the upper limit is not particularly limited to, and a buffer solution with a concentration in the range from about 0.005 M to about 1.5 M, from about 0.075 M to about 1.0 M, or from about 0.1 M to about 0.5 M can be employed.
  • the ion exchanger is selected by taking into consideration the isoelectric point of the protein (target protein) to be separated.
  • the target protein is adsorbed under a condition for allowing the target protein to be positively charged, or at a lower pH than the isoelectric point of the target protein (low pH condition), for ensuring adsorption of the target protein on the ion exchanger when a cation exchanger is used.
  • the low pH condition differs depending on the isoelectric point of the target protein, and the pH may be adjusted in the range from 4.0 to 4.3, for example, when the isoelectric point of the target protein is at pH 4.5.
  • the pH is adjusted so that all the proteins are positively charged.
  • the proteins may be adsorbed, for example, in the pH range from 4.0 to 4.1.
  • the protein is adsorbed under a condition for allowing the protein to be negatively charged, or at a higher pH (low pH condition) than the isoelectric point of the target protein when an anion exchanger is used.
  • the target protein can be adsorbed on the ion exchanger as described above by selecting a condition in which the pH is not in the vicinity of the isoelectric point of the target protein.
  • the pH for adsorption it is preferable for determining the pH for adsorption to take pH stability of the target protein into consideration.
  • the pH is preferably controlled in the range not deactivating the protein or decreasing the activity of the protein, when the target protein is required to be separated while it maintains the activity.
  • the kind of the ion exchanger is not particularly limited to.
  • the ion exchanger (resin) available include styrene, acrylic, methacrylic, phenol, aliphatic, pyridine, dextran or cellulose base reins into which cationic, anionic or amphoteric ion exchange groups are introduced.
  • carboxylic groups (CM) or sulfuric groups (SM and Mono S) are used as the cationic ion exchange group
  • amino groups (DEAE, QAE and Mono Q) are used as the anionic ion exchange groups.
  • a variety of ion exchangers are commercially available (for example those sold by Seikagaku Co., Amersham Biosciences K.K.), and commercially available ion exchangers providing appropriate characteristics may be used in the invention.
  • the buffer solution used for allowing the sample to adsorb preferably comprises a combination of a weak acid and weak base (such as tris-succinate buffer solution, tris-citric acid buffer solution and tris-acetic acid buffer solution).
  • a buffer solution used in the elution condition (second condition) described below to be prepared by decreasing the concentration of the acid or base constituting the buffer solution or, in other words, a buffer solution having low ion strength and at a pH closer to the isoelectric point side of the target protein than in the first condition can be prepared. Accordingly, there is no need to prepare plural kinds of buffer solutions having different combinations of the acid and base. The entire operation is further simplified since shift from adsorption (and washing) to elution is possible by merely changing the proportion of the acid or base in the buffer solution, and a chain of continuous operations is possible.
  • the same buffer solution as used in adsorption may be usually used for equilibration.
  • An elution step (step b) follows step a.
  • the elution step is performed under the second condition at a lower ion strength than in the first condition, and at a pH closer to the isoelectric point side of the target protein than in the first condition. Since the method of the invention is featured in that the ion strength in the second condition is adjusted to be lower than the ion strength in the first condition and the protein is adsorbed at a high ion strength and eluted at a low ion strength, the method is effective for specifically separating the target protein (in particular glycoprotein). In other words, lowering the ion strength for elution prevents unnecessary components adsorbed on the ion exchanger from being eluted, or the target protein can be selectively and efficiently eluted.
  • the ion strength in the second condition may be determined by taking other conditions (such as the kind of the target protein, the kind of the sample containing the protein, or the kind of the buffer solution) into consideration, with the proviso that it is essential that the ion strength is lower than the ion strength in the first condition.
  • the second condition is determined so that the concentration of the weak acid or base is lower than the concentration in the first condition within the range from about 0.05 M to about 0.5 M, preferably from about 0.075 M to about 0.1 M, and further preferably from about 0.1 M to about 0.75 M.
  • the pH in the second condition is adjusted to be closer to the isoelectric point side of the target protein than in the first condition.
  • the pH in the second condition is adjusted to be in the vicinity of or higher than the isoelectric point of the target protein.
  • the pH in the second condition is adjusted to be in the vicinity of or lower than the isoelectric point of the target protein.
  • An elution solution satisfying the above-mentioned second condition is prepared in the elution step, and the ion exchanger after the adsorption step is made to contact the elution solution.
  • the sample subjected to the adsorption step contains plural kinds of the target proteins, these plural proteins can be continuously eluted by continuous elution at solvent conditions corresponding to respective isoelectric points of the target proteins.
  • a buffer solution having a quite different composition from the buffer solution used in the adsorption step may be used, it is preferable to use a buffer solution obtained by reducing the concentration of either the acid or the base in the buffer solution used in the adsorption step (the other concentration is preferably maintained as before).
  • a buffer solution comprising a combination of the same acid and the same base as in the buffer solution used in the adsorption step is used in the elution step in the above-mentioned aspect.
  • one of the features of the second condition, or a low ion strength, as well as the other feature, or a pH closer to the isoelectric point side of the target protein than in the first condition may be simultaneously satisfied in this aspect.
  • the ion strength is lowered by reducing the concentration of the acid constituting the buffer solution.
  • the pH shifts to a pH closer to the isoelectric point side of the target protein (shift to higher pH), and the second condition is satisfied.
  • the pH of the buffer solution used in the adsorption step is higher than the isoelectric point of the target protein, the ion strength is lowered by reducing the concentration of the base constituting the buffer solution.
  • the pH shifts to a pH closer to the isoelectric point side of the target protein (shift to lower pH), and the second condition is satisfied.
  • the aspect for permitting the concentrations of the acid or base to change in the buffer solution used reduction of the ion strength and adjustment of the pH may be simultaneously achieved by simple operations. These aspects are particularly effective when a plurality of proteins or glycoproteins having different isoelectric points is present in the sample, and when each of these proteins are to be separated or fractionated, since continuous reduction of the ion strength and control of the pH are also possible by simple operations.
  • a washing step (step c) is preferably interposed between the adsorption step (step a) and elution step (step b).
  • the object of the washing step is to remove unnecessary components (components other than the target protein in the sample) adsorbed on the ion exchanger after the adsorption step by washing.
  • the washing solution used preferably satisfies a condition by which the unnecessary components are effectively removed while the target protein adsorbed on the ion exchanger is not eluted.
  • the ion exchanger is washed in the washing step with a buffer solution having the same composition as the buffer solution used in the adsorption step (or substantially under the same condition).
  • the composition of the buffer solution may be slightly changed for enhancing the effect of washing.
  • a buffer solution to which a small amount of appropriate salts are added may be used as the washing solution. It is preferable to wash the ion exchanger with a sufficient amount of the washing solution in order to attain a good washing effect. For example, when the ion exchanger packed in a column is used, the column is washed using the washing solution twice to 30 timed by volume, preferably 3 to 20 times by volume, of the volume of the ion exchanger.
  • the temperatures of a series of operations are usually room temperature or a low temperature (for example in the range from 4 to 10° C.).
  • the separation method of this aspect comprises following steps (a) and (b):
  • Step (a) is performed under a condition (first condition) at the first ion strength and at a pH outside of the vicinity of the isoelectric point of the target protein.
  • a relatively low ion strength is employed as the first ion strength, since this condition affords a favorable condition for permitting a protein (or glycoprotein) having weak adsorption ability to the support to be adsorbed.
  • the separation method in this aspect is usually utilized for separation and recovery of the protein (or glycoprotein) having weak adsorption ability to the support.
  • the concentration is, for example, about 0.08 M or less, preferably about 0.05 M or less, more preferably about 0.03 M or less and further preferably about 0.02 M or less when a buffer solution comprising a weak acid and weak base is used.
  • the lower limit is not particularly limited to, and a buffer solution with a concentration in the range from about 0.001 M to about 0.08 M, from about 0.01 M to about 0.05 M or from about 0.02 M to about 0.03 M may be used.
  • the elution step (step b) follows the step (a).
  • This elution step is preformed under the second condition at an ion strength of equal to or lower than the ion strength in the first condition, and at a pH closer to the isoelectric point of the target protein than in the first condition.
  • the ion strength in the second condition is determined to be equal or lower than the ion strength in the first condition.
  • the method of the invention is featured in that a higher ion strength is not used for elution, and is effective for specifically separating the target protein (particularly the glycoprotein). Selecting a low ion strength for elution permits unnecessary components adsorbed on the ion exchanger to be prevented from being eluted. In other words, the target protein can be specifically and efficiently eluted.
  • the ion strength in the second condition may be determined by taking other conditions (the kind of the target protein, the kind of the sample containing the protein, or the kind of the buffer solution used) into consideration under an essential condition that the ion strength in the second condition is equal to or lower than the ion strength in the first condition.
  • the protein is eluted at an ion strength about 1 to 5 times of the ion strength for adsorption (for example, the concentration of the buffer solution is about 0.02 M for adsorption and about 0.02 to about 0.1 M for elution).
  • the protein may be eluted at an ion strength preferably about 1 to 3 times, more preferably about 1 to 1.5 times, and most preferably about 1 to 1.2 time of the ion strength for adsorption.
  • the ion strength in the second condition may be determined so as to be equal or lower than in the first condition.
  • DAO diamine oxidase
  • SP-Sephadex c-50 (Amersham Biosciences K.K. 2.5 ⁇ 6 cm), a cation resin, was equilibrated with 0.1 M Tris succinate buffer, pH 6.5. Pea epicotyls were homogenized with 0.2 M Tris succinate buffer, pH 7.5. The homogenate was squeezed through a nylon mesh (74 ⁇ m) to remove insoluble substances. The solution was brought to pH 6.5 with saturated succinic acid and to twice the volume of the extraction buffer with distilled water, and then centrifuged at 15000 g for 20 min. (Step 1: crude extract) Next, the crude extract (the supernatant of centrifugation) was applied to the SP-Sephedex c-50 column.
  • FIG. 1 shows the results of chromatography performed as described above.
  • the horizontal axis is the fraction number (time axis) and the spindle axis is OD at 280 nm (left), and DAO activity (right).
  • the peaks for OD280 and DAO activity are in the vicinity of fraction number 5 .
  • Active fractions (3-11) were gathered and used as a purified enzyme (Step 2: Final purification).
  • DAO activity, purification and yield are summarized in the Table in FIG. 2 .
  • the purified enzyme had a specific activity of 1.38 ⁇ kat/mg protein with purification of 115 and this was almost the same as the value reported in the literature. The yield with this procedure (88.7%) is much greater than that with the previous method.
  • the purified enzyme was subjected to SDS electrophoresis. As a result, a single band was seen with silver staining (result not shown).
  • DAO Diamine oxidase
  • Pea. McGuil et al. established a method for its purification in 1994. The method involved crude extract, ammonium sulfate precipitation, organic solvent precipitation, DEAE-cellulose column chromatography, gel filtration, and hydroxyapatite column chromatography. These are general purification techniques that gradually remove contaminating proteins through repeated precipitation and dialysis, and take advantage of the electric properties of proteins and differences in molecular weights. These techniques usually require 3-4 days.
  • the present simple purification method which includes cation exchange and Tris succinate buffer, enabled us to purify DAO to homogeneity by silver staining in 12 hours or less while simultaneously achieving a higher yield than the previous method.
  • CM-Sephadex C-50 (Amersham Biosciences K.K. 1.6 ⁇ 6 cm), a Cation Resin, was Equilibrated with 0.1 M Tris Succinate Buffer, pH 4.0.
  • the volume of egg white was measured and five volumes of distilled water were added. This mixture was stirred for 30 min. To 20 ml of this solution was added 50 ml of 0.2 M Tris succinate buffer, pH 7.5, and the pH was adjusted to 4.0 with saturated succinic acid. In addition, this solution was brought to 100 mL with distilled water. (Ovomucin can be efficiently removed from egg white by dilution with distilled water two or three times, or by precipitation at pH 4.0.) The solution was then centrifuged at 15000 g for 20 min, and 10 mL of the supernatant was applied to the column.
  • FIG. 3 shows the results of chromatography performed as described above.
  • the horizontal axis is the fraction number (time axis), and the spindle axis is OD at 280 nm.
  • the peak of 280 nm was located in fraction numbers 22 - 36 .
  • Ovalbumin (isoelectric point pH4.5) could be efficiently collected from egg white in one step.
  • FIG. 5 shows the results of chromatography performed as described above.
  • the horizontal axis is the fraction number (time axis) and the spindle axis is OD at 280 nm.
  • the peak of 280 nm is in the vicinity of fraction numbers 14 - 19 , which were gathered and used as a final purified protein.
  • FIG. 6 shows the results of protein silver staining by SDS-PAGE. The only band of ovalbumin was confirmed in fraction numbers 14 - 19 . Upon washing with 0.1 M Tris (about pH 9.5), although absorption of OD280 nm was noted in fraction numbers 32 - 35 , a band of ovalbumin was not confirmed by SDS-PAGE.
  • the elution conditions and the results of purifications 1 and 2 of ovalbumin are reference points for solving the problem of how to most efficiently collect a target protein.
  • the pH of the buffer in the ovalbumin-elution step is slightly different between purifications 1 and 2 .
  • purification 1 buffer with the same pH (4.5) as the isoelectric point of ovalbumin is used as the buffer for elution.
  • the isoelectric point of ovalbumin (pH 4.5) is between the pH (4.75) of the buffer solution used for elution and the pH (4.0) of the buffer solution used for adsorption and washing.
  • ovalbumin isoelectric point pH4.5, molecular weight 45 k, Sigma-Aldrich Corporation
  • transferrin isoelectric point pH6.2, molecular weight 80 k, Sigma-Aldrich Corporation
  • streptavidin isoelectric point of total protein pH6.4, isoelectric point of the surface pH5.0-5.5, molecular weight 60 k, tetramer, GR, Nacalai Tesque Inc.
  • CM-Sephadex C-50, CM-Sepharose FastFlow, SP-Sephadex C-50, and SP-Sepharose FastFlow All from Amersham, Biosciences K.K.
  • Samples were added to the columns (0.1M Tris succinate buffer pH4.0) and washed (0.1M Tris succinate buffer pH4.0) according to the procedure in example 1. Elution was performed with a gradient of from ca pH4.0 to ca pH9.0, generated by a continuous decrease in the succinate concentration.
  • FIGS. 7-10 The results of chromatography with CM-Sephadex, CM-Sepharose, SP-Sephadex, and SP Sepharose are shown in FIGS. 7-10 , respectively.
  • the horizontal axis is the fraction number (time axis)
  • the spindle axis is OD at 280 nm (left) and the pH of the elution solution (right).
  • the peak of ovalbumin is in the neighborhood of fraction number 26
  • the peak of streptavidin is in the neighborhood of fraction number 37
  • the peak of transferrin is in the neighborhood of fraction number 39 .
  • the peak of ovalbumin is in the neighborhood of fraction number 16
  • the peak of streptavidin is in the neighborhood of fraction number 35
  • the peak of transferrin is in the neighborhood of fraction number 38
  • the peak of ovalbumin is in the neighborhood of fraction number 13
  • the peak of streptavidin is in the neighborhood of fraction number 28
  • the peak of transferrin is in the neighborhood of fraction number 37 .
  • ovalbumin isoelectric point pH4.5, molecular weight 45 k, Sigma-Aldrich Corporation
  • transferrin isoelectric point pH6.2, molecular weight 80 k, Sigma-Aldrich Corporation
  • streptavidin isoelectric point of total protein pH6.4, isoelectric point of the surface pH5.0-5.5, molecular weight 60 k, tetramer, GR, Nacalai Tesque Inc.
  • CM-Sephadex was used as an ion-exchanger.
  • FIG. 11 shows the results of chromatography.
  • the horizontal axis is the fraction number (time axis) and the spindle axis is OD at 280 nm.
  • Ovalbumin shows a peak in the neighborhood of fraction number 16 and transferrin shows a peak in the neighborhood of fraction number 38 .
  • the non-glycoprotein streptavidin did not adsorb to the ion-exchanger.
  • the simple purification method of the invention is effective for separating glycoproteins.
  • glycoproteins showed a stronger adsorption tendency compared with the non-glycoprotein. Therefore, if the ion strength is set high enough so that a non-glycoprotein can not adsorb to the ion-exchanger, a glycoprotein can be adsorbed specifically. In other words, when adsorption is performed under the conditions that non-glycoproteins can not adsorb to the ion-exchanger, a target glycoprotein can be purified from a sample containing non-glycoproteins mixed together.
  • glycoproteins that had adsorbed to the ion-exchanger continued to flow gradually upon washing for a long time with a high-concentration buffer. It was showed that the difference in adsorbability makes a difference in the effect of washing.
  • Carbonic anhydrase (Sigma c7025) and peroxidase (Wako Pure Chemicals 309-50993) were adsorbed to CM-Sephadex as a cation-exchanger using 20 mM Tris succinate buffer (pH 4.5). Elution was performed with a linear gradient (from ca pH4.5 to ca pH8.5) by adding 20 mM Tris after washing.
  • FIG. 13 shows the results of chromatography.
  • Fraction numbers 29 - 35 and 39 - 47 show the elution peaks of carbonic anhydrase and peroxidase, respectively.
  • This method makes it possible to separate a glycoprotein and a common protein with an exceptionally weak bond under high ion strength because adsorption occurs under a low ion strength.
  • this method could be used to separate proteins which are not adsorbed onto the column under high ion strength (under a high buffer concentration), by adjusting the concentration etc. of the buffer.
  • Tris acetate buffer and Tris citrate buffer were examined.
  • Tris acetate buffer pH4.0 was used for adsorption and washing, and a gradient of from ca pH4.0 to ca pH 9.0, generated by a continuous decrease in the acetic acid concentration, was used for elution (condition 1 ).
  • Tris citrate buffer pH4.0 was also used for adsorption and washing, and a gradient o from ca pH4.0 to ca pH 9.0 generated by a continuous decrease in the citric acid concentration, was used for elution (condition 2 ). Ovalbumin, transferrin, and streptavidin were used for the sample. CM-Sephadex was used as an ion-exchanger.
  • FIGS. 14 and 15 The results of chromatography under conditions 1 and 2 are shown in FIGS. 14 and 15 , respectively.
  • the horizontal axis is the fraction number (time axis) and the spindle axis is OD at 280 nm (left) and the pH of the eluate (right).
  • the peak of all proteins applied to the column is in the vicinity of fraction number 32 .
  • the peak of all proteins applied to the column is in the vicinity of fraction number 27 .
  • an appropriate buffer can be selected based on a consideration of the intended use of the target protein after extraction and the isoelectric point of the target glycoprotein, the isoelectric point of other coexisting glycoproteins and non-glycoproteins.
  • the method of the invention can be used to quickly and efficiently collect a target protein or glycoprotein from an incoherent sample that contains many kinds of protein and glycoprotein. Moreover, all of the glycoproteins that were subjected to examination could be collected, and in the glycoprotein, reversible adsorption was seen under a high ion strength. Therefore, this method shows high generality for the separation of glycoproteins.
  • the invention may be applied for various objects as the methods for separating and purifying proteins.
  • the invention is applicable for separation and recovery of useful proteins (disease-related glycoproteins) from biological samples (for example serum), and for detecting specified proteins (for example, a specified glycoprotein is compared between normal persons and patients to use the results for diagnosis).
  • useful proteins disease-related glycoproteins
  • specified proteins for example, a specified glycoprotein is compared between normal persons and patients to use the results for diagnosis.
  • the invention may be used as a separation method when proteins or glycoproteins should be eliminated such as production of allergy-free vaccines.
  • the invention may be also used for detecting or recognizing isoelectric points of various proteins.
  • the invention is also useful for separating and purifying a target glycoprotein from a sample containing foreign proteins being different only in the presence or absence of sugar chains.
  • the invention may be advantageous in separation and purification of the glycoprotein, the invention can be also applied for efficiently separating common proteins as well as various glycoproteins by combining the buffer solutions and columns.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
US10/594,441 2004-03-30 2004-11-08 Method of Separating Protein Abandoned US20080269469A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2004098022 2004-03-30
JP2004-098022 2004-03-30
PCT/JP2004/016549 WO2005100379A1 (fr) 2004-03-30 2004-11-08 Procédé de séparation d'une protéine

Publications (1)

Publication Number Publication Date
US20080269469A1 true US20080269469A1 (en) 2008-10-30

Family

ID=35149942

Family Applications (1)

Application Number Title Priority Date Filing Date
US10/594,441 Abandoned US20080269469A1 (en) 2004-03-30 2004-11-08 Method of Separating Protein

Country Status (4)

Country Link
US (1) US20080269469A1 (fr)
EP (1) EP1739089A4 (fr)
JP (1) JP3920913B2 (fr)
WO (1) WO2005100379A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009178088A (ja) * 2008-01-30 2009-08-13 Aichi Univ Of Education ポリアミンオキシダーゼの精製法
JPWO2022239704A1 (fr) 2021-05-10 2022-11-17

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070144973A1 (en) * 2003-05-16 2007-06-28 Cryobiophysica, Inc. External gradient chromatofocusing

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITBO20010426A1 (it) * 2001-07-06 2003-01-06 Alfa Wassermann Spa Processo per la purificazione di proteine farmacologicamente attive mediante cromatografia in scambio cationico

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070144973A1 (en) * 2003-05-16 2007-06-28 Cryobiophysica, Inc. External gradient chromatofocusing

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9488625B2 (en) 2010-12-15 2016-11-08 Baxalta GmbH Purification of factor VIII using a conductivity gradient

Also Published As

Publication number Publication date
JP3920913B2 (ja) 2007-05-30
JPWO2005100379A1 (ja) 2008-03-06
EP1739089A4 (fr) 2007-05-02
EP1739089A1 (fr) 2007-01-03
WO2005100379A1 (fr) 2005-10-27

Similar Documents

Publication Publication Date Title
US5169936A (en) Protein purification on immobilized metal affinity resins effected by elution using a weak ligand
JP6593721B2 (ja) 高純度の組換えヒト血清アルブミンを単離、精製するクロマトグラフ法
US5252709A (en) Chromatographic separation of plasma proteins
CN103998469B (zh) 抗体纯化方法
CN105153297B (zh) 一种从Cohn组分Ⅳ沉淀中分离纯化α2-巨球蛋白的方法
US10981975B2 (en) Method for efficient purification of human serum albumin
EP2102335B1 (fr) Purification de facteur xi
CA2059979C (fr) Complexe contenant le facteur de coagulation ix
Josić et al. Isolation of plasma proteins from the clotting cascade by heparin affinity chromatography
AU2005297061B2 (en) Method for the isolation of haptoglobin
KR20150033739A (ko) 알부민의 연마 방법
US20080269469A1 (en) Method of Separating Protein
Gunzer et al. Purification of α1-proteinase inhibitor by triazine dye affinity chromatography, ion-exchange chromatography and gel filtration on fractogel TSK
Fweja et al. Isolation of lactoperoxidase using different cation exchange resins by batch and column procedures
KR100361894B1 (ko) 치환크로마토그라피방법및정제된헤모글로빈생성물
KR100730902B1 (ko) 글리코실화된 단백질과 비글리코실화된 단백질을 분리하는방법
JP2022505307A (ja) Ｃ１－ｉｎｈを精製する方法
US20230143163A1 (en) Protein a chromatography purification of an antibody
US20220348640A1 (en) Elution of monoclonal antibodies in protein a affinity chromatography
JP5458299B2 (ja) 甘味誘導物質ミラクリンの精製方法
SU962302A1 (ru) Способ одновременной очистки протеолитического фермента и его обратимого ингибитора
EP0475311A1 (fr) Procédé d'enlèvement des protéines originaires d'Escherichia coli
JP2022538355A (ja) Ｃ１－ｉｎｈを精製する方法
Tadepalli et al. Separation of protein C from fraction IV of the Cohn process using immobilized metal affinity chromatography
JPH11287792A (ja) インターロイキン６レセプターの精製法

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION